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Abstract Aim to current situation of urgent need to develop a simple, rapid, sensitive and specific technique for White spot syndrome virus (WSSV) detection in shrimp (Penacus orientalis) culture industry. Six specific primers including one set of outer primeres F3 and B3, one set of inner primers FIP and BIP, and one set of loop primers LF and LB were targeted on the conserved region in ORF120 of WSSV genome. These primers were designed and synthesized for amplifying the target gene ORF120 of WSSV in a loop mediated isothermal amplification (LAMP) system. A visual WSSV-LAMP was developed based on the optimized reaction conditions in which calcein was applied to be detection indicator. As none amplification taken place in the detection, calcein was cancellation by chelated with manganese chloride. When WSSV target gene was existed in the detected sample, a large amount of DNA was synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combined with metallic ion competitively to form an insoluble pyrophosphate salt. Calcein was emitted visual Kelly fluorescence. It took about 60 min to finish the detection during the one-step amplification reaction. Testing results showed that this WSSV-LAMP assay was able to detect the target gene contained in WSSV positive samples by nake-eye observation through the Kelly fluorescence emited by the amplification production. The lowest detected limitation was 1fg of target gene DNA. The preliminary tested results of clinical application showed that fifteen positive samples with same labels were figured out from 250 shrimp clinical samples by WSSV-LAMP and WSSV-PCR recommened by Office International Des Epizooties, 2012, respectively. The diagnose accordance rate between WSSV-LAMP and WSSV-PCR was hundred percent.WSSV-LAMP could also detected another three samples as positive, which were confirmed negative by WSSV-PCR. The comparison between these two methods, WSSV-LAMP was higher positive detection rate than that of WSSV-PCR , in which WSSV-LAMP was 7.2%(18/250) and WSSV-PCR was 6.0%(15/250). Results suggest that WSSV-LAMP is with higher technical advantage than WSSV-PCR in the detection of clinical samples with lower virus quantities. It can be used as an on-the-spot detection tool for WSSV in clinical diagnosis, breeding and port quarantine inspection,as well as sea ecological monitoring.
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Received: 20 December 2012
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